HIV-1, a human lentivirus, is the major causative agent of AIDS, which presently infects approximately 42 million persons worldwide with 1 million infected persons in North America.
Although considerable effort is being put into the design of effective therapeutics, currently available drugs are not effective to cure AIDS. In attempts to develop such drugs, several stages of the HIV-1 life cycle have been considered as targets for therapeutic intervention (H. Mitsuya, et al., FASEB J., 1991, 5, 2369-81). Many viral targets for intervention with HIV-1 life cycle have been suggested. A schematic illustration of the life-cycle of HIV-1, showing some of the potential drug targets for the treatment of HIV-1 infection and AIDS is illustrated in FIG. 1. Most of the currently available treatments inhibit reverse transciptase enzyme or the HIV-1 protease enzyme. Recently, enfuvirtide, a drug with a new mechanism of action was approved. Enfuvirtide is a peptide that binds to a region of the envelope glycoprotein 41 of HIV-1 that is involved in the fusion of the virus with the membrane of the CD4 positive host cell, and thereby inhibits fusion of HIV-1 with the membrane of the CD4 positive cells (J. P. Lalezari, et al., “Enfurvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America”, N. Engl. J. Med., 2003, 348, 2175-85).
The new treatment regimens for HIV-1 show that a combination of anti-HIV compounds, which target reverse transcriptase (RT), such as azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddI), dideoxycytidine (ddC) used in combination with an HIV-1 protease inhibitor have a far greater effect (2 to 3 logs reduction) on viral load compared to AZT alone (about 1 log reduction). One such example is a combination of AZT, ddI, 3TC and ritonavir (A. S. Perelson, et al., Science, 1996, 15:1582-86).
Table 1 lists the drugs currently approved by the United States Food and Drug Administration for the treatment of HIV infection and AIDS.
TABLE 1United-States Food and Drug Administration ApprovedDrugs Treatment of HIV-1 Infection and AIDSDrug ClassDrugNuceloside reverse transcriptase lamivudine and zidovudineinhibitorNuceloside reverse transcriptase emtricitabine, FTCinhibitorNuceloside reverse transcriptase lamivudine, 3TCinhibitorNuceloside reverse transcriptase abacavir and lamivudineinhibitorNuceloside reverse transcriptase zalcitabine, dideoxycytidine, ddCinhibitorNuceloside reverse transcriptase zidovudine, azidothymidine, AZT, ZDVinhibitorNuceloside reverse transcriptase abacavir, zidovudine, and lamivudineinhibitorNuceloside reverse transcriptase tenofovir disoproxil fumarate and inhibitoremtricitabineNuceloside reverse transcriptase enteric coated didanosine, ddI ECinhibitorNuceloside reverse transcriptase didanosine, dideoxyinosine, ddIinhibitorNuceloside reverse transcriptase tenofovir disoproxil fumarate, TDFinhibitorNuceloside reverse transcriptase stavudine, d4TinhibitorNuceloside reverse transcriptase abacavir sulfate, ABCinhibitorNon-nuceloside reverse delavirdine, DLVtranscriptase inhibitorNon-nuceloside reverse efavirenz, EFVtranscriptase inhibitorNon-nuceloside reverse nevirapine, NVPtranscriptase inhibitorProtease inhibitoramprenavir, APVProtease inhibitortipranavir, TPVProtease inhibitorindinavir, IDV,Protease inhibitorsaquinavir mesylate, SQVProtease inhibitorlopinavir and ritonavir, LPV/RTVProtease inhibitorFosamprenavir Calcium, FOS-APVProtease inhibitorritonavir, RTVProtease inhibitordarunavirProtease inhibitoratazanavir sulfate, ATVProtease inhibitornelfinavir mesylate, NFVFusion inhibitorEnfuviritide, T-20Multi-class combinationefavirenz, emtricitabine and tenofovir disoproxil fumarate
In spite of these advances, the need remains for new drugs that are effective against HIV-1 infection and AIDS. It is possible that long-term use of combinations of these chemicals that comprise the currently available treatments will lead to toxicity, especially to the bone marrow. Long-term cytotoxic therapy may also lead to suppression of CD8 positive T cells, which are essential to the control of HIV, via killer cell activity (V. Blazevic, et al., AIDS Res. Hum. Retroviruses, 1995, 11, 1335-42) and by the release of suppressive factors, notably the chemokines Rantes, MIP-1α and MMIP-1β (F. Cocchi, et al., Science, 1995, 270, 1811-1815). Another major concern in long-term chemical anti-retroviral therapy is the development of HIV mutations with partial or complete resistance (J. M. Lange, AIDS Res. Hum. Retroviruses, 1995, 10, S77-82). Such mutations may be an inevitable consequence of anti-viral therapy. The pattern of disappearance of wild-type virus and appearance of mutant virus due to treatment, combined with coincidental decline in CD4 positive T cell numbers strongly suggests that, at least with some compounds, the appearance of viral mutants is a major underlying factor in the failure of AIDS therapy.
The HIV-1 virus contains a 10-kb single-stranded, positive-sense RNA genome that encodes three major classes of gene products that include: (i) structural proteins such as Gag, Pol and Env; (ii) essential trans-acting proteins (Tat, Rev); and (ii) “auxiliary” proteins that are not required for efficient virus replication in at least some cell culture systems (Vpr, Vif, Vpu, Nef).
One approach to treating individuals infected with HIV-1 is to administer to such individuals compounds that directly intervene in and interfere with the machinery by which HIV-1 replicates itself within human cells.
One such protein, Vif (viral infectivity factor), is expressed by all known lentiviruses except equine infectious anemia virus. Vif protein of HIV-1 is a highly basic, 23-kDa protein composed of 192 amino acids. Sequence analysis of viral DNA from HIV-1-infected-individuals has revealed that the open reading frame of Vif remains intact. (P. Sova, et al., J. Virol. 1995, 69, 2557-64; U. Wieland, et al., Virology, 1994, 203, 43-51; U. Wieland, et al., J. Gen. Virol., 1997, 78, 393-400). Vif is required for efficient virus replication in vivo, as well as in certain host cell types in vitro because of its ability to overcome the action of a cellular antiviral system. Deletion of the Vif gene dramatically decreases the replication of simian immunodeficiency virus (SIV) in macaques and HIV-1 replication in SCID-hu mice (G. M. Aldrovandi, et al., J. Virol., 1996, 70, 1505-11; R. C. Desrosiers, et al., J. Virol., 1998, 72, 1431-37), indicating that the Vif gene is essential for the pathogenic replication of lentiviruses in vivo.
Recent studies have elucidated the mechanism underlying the importance of Vif to the replication of HIV-1 viruses in vivo, which is associated with the anti-viral effect of a host protein called hA3G (APOBEC3G) (CEM15). hA3G belongs to a family of cytidine deaminases which induces G to A hypermutation in newly synthesized viral cDNA. Packaging of hA3G into virus particles can result in hypermutation of the viral minus-strand cDNA during reverse transcription, thereby interfering with the replication of the virus. R. S. Harris, et al., Cell, 2003, 113, 803-09; B. Mangeat, et al., Nature, 2003, 424, 99-103. Consistent with the antiviral effect of hA3G, correlations have been observed between hA3G mRNA levels and HIV viral load and CD4 cell count, both of which are predictors of HIV disease progression in patients who have not received antiretroviral drugs or other forms of therapeutic intervention. In addition, it was found that HIV-infected patients who show a low rate of disease progression even in the absence of antiviral treatment (“long term non-progressors”) have significantly higher hA3G mRNA levels than do HIV-uninfected controls or the progressors, whose hA3G mRNA levels are significantly lower that of HIV-uninfected controls. X. Jin, et al., J. Virol., 2005, 79(17), 11513-16.
The importance of Vif to HIV-1 replication is believed to be due to its role in overcoming the host defense mechanism provided by hA3G. Vif counteracts the antiviral activity of hA3G by targeting it for destruction by the ubiquitin-proteasome pathway. Vif forms a complex with hA3G and enhances hA3G ubiquitination, thereby targeting hA3G for degradation via the ubiquitin-proteasome pathway. A. Mehle, et al., J. Biol. Chem., 2004, 279(9), 7792-98. B. R. Cullen, J. Virol. 2006, 80, 1067-76.
While the detailed molecular mechanism of the effect of Vif in evading host cell defense to HIV-1 remains to be elucidated, it has been hypothesized that Vif self-association to form multimers may play a key role, and the multimerization of Vif has been found to be required for Vif function in the viral life cycle. S. Yang, et al., J. Biol. Chem., 2001, 276(7), 4889-93. It has been demonstrated that Vif aggregation is not simply due to fortuitous aggregation, but that a specific domain affecting Vif self-association is located at the C terminus of this protein, especially the proline-enriched 151-164 region, is implicated in Vif multimerization.
In cell culture systems, Vif-deficient (Vif−) HIV-1 is incapable of establishing infection in certain cells, such as H9 T cells, peripheral blood mononuclear cells, and monocyte-derived macrophages. This has led to classification of these cells as nonpermissive. In other cells, the Vif gene is not required; these cells have been classified as permissive. Using this phenomenon, Yang, et al., demonstrated that Vif aggregation (multimerization) is essential to the role of Vif in promoting viral infectivity. Yang, et al. tested various Vif mutants in a modified single-round viral infectivity assay. Wild-type Vif or its mutants were expressed in the nonpermissive H9 T-cells. At the same time, pseudotyped (with VSV envelope) HIV-1 viruses, without vif and env in their genome, were generated from these cells. The recombinant viruses were allowed to infect target cells harboring an expression cassette containing the HIV-1 long terminal repeat promoter-driven CAT gene. The viral infectivity was measured by the level of CAT gene expression in the target cells. When the wild-type Vif gene was expressed in the Vif-defective HIV-1 virus-producing nonpermissive H9 T-cells, a high level of viral infectivity was observed. However, when a Vif mutant lacking the binding region (VifΔ151-164) was expressed in the Vif-defective HIV-1 virus-producing nonpermissive H9 T-cells, the viral infectivity was almost unaltered, compared with the Vif-defective HIV-1 viruses, indicating that the deletion severely decreased the function of Vif protein and made it unable to rescue the infectivity of the Vif-defective HIV-1 viruses generated from nonpermissive T-cells. This experiment demonstrated that multimerization of Vif proteins is required for Vif function. S. Yang, et al., J. Biol. Chem., 2001, 276(7), 4889-93. A scanning mutational analysis of Vif also demonstrated the importance of the binding region to infectivity, particularly the three residues (PPL) at 161-163. These could be substituted individually without loss of function, but substitution of all three residues severely inhibited function. J. H. M. Simon, et al., J. Virol, 1999, 73(4), 2675-81.
The discovery that multimerization of Vif proteins is required for Vif function in the viral life cycle, has led to it being proposed as a potential novel target for anti-HIV-1 therapeutics. The hypothesis is illustrated in FIGS. 2A and 2B. As shown by the above-mentioned studies, Vif multimerization is believed to be essential to Vif's action in an HIV-1 infected cell. When Vif functions normally in the cytoplasm, it is believed that Vif dimerizes and that the resulting Vif dimers target hA3G for modification through ubiquitination; and that as a consequence the hA3G is destroyed by the proteosome, and the HIV life cycle is not interrupted. Thus, when Vif functions normally, the virus overcomes the anti-infective function of hA3G. However, when Vif function is compromised (e.g. through the action of a drug that inhibits Vif dimerization, although HIV-1 would still be able to bind CD4 and enter T-cells, the hA3G would hypermutates the viral DNA during reverse transcription resulting in mutated viral DNAs that are either destroyed or integrated in defective form into the chromosomal DNA. If the mutated DNA is transcribed, the resulting RNA would encode few or no functional proteins and most of the HIV-1 viruses produced by the cell would be defective and non-infective.
Yang, et al. identified peptides containing a PXP motif that bind to HIV-1 Vif protein. These proline-enriched peptides were found to inhibit the Vif-Vif interaction in vitro. In addition, peptides covering all the amino acids of the HIV-1 Vif protein sequence were prepared and it was found that proline-enriched peptides that contain the 161PPLP164 domain were able to inhibit the Vif-Vif interaction. The study concluded that the proline-enriched peptides block the multimerization of Vif through interfering with the polyproline interfaces of Vif formed by the 161PPLP164 domain. Moreover, these peptides which inhibit the Vif-Vif interaction in vitro were found to inhibit HIV-1 replication in the “nonpermissive” T-cells. B. Yang, et al., J. Biol. Chem., 2003, 278(8), 6596-602.